摘要
为了给表达高活性的抗菌肽Hadrurin提供开发应用的依据,进一步为用基因工程方法生产具有多种生物活性的抗菌肽Hadrurin蛋白提供研究基础,本研究表达获得重组抗菌肽Hadrurin(rHadrurin)蛋白的基础上,将纯化的rHadrurin蛋白进行肠激酶酶切,恢复其天然活性结构。用最小抑菌浓度(MIC)和最小杀菌浓度(MBC)方法检测抗菌肽Hadrurin在不同剂量、不同pH值、不同保存温度下对鸡致病性大肠杆菌和金黄色葡萄球菌的抑菌活性及杀菌活性。并以小鼠为动物模型,进行抗菌肽rHadrurind对小鼠的体内保护实验。结果表明抗菌肽rHadrurin对上述细菌的最小抑菌范围为1.32μg/mL～4.32μg/mL,最小杀菌范围为1.77μg/mL～8.54μg/mL,而且-70℃～100℃及在pH3～pH10条件下仍具有高效抗菌活性。240μg/只剂量的抗菌肽蛋白可有效预防保护小鼠免受致死剂量鸡致病性大肠杆菌攻击,320μg/只剂量可达到保护率85%以上。
Hadrurin was originally found in the venom of the Mexican scorpion Hadrurus aztecus and has demonstraed antimicrobial activity against a wide range of bacteria. In this study,Hadrurin was expressed in E.coli BL21 from the recombinant expression plasmid pET-H. The protein was purified by affinity chromatography,and digested with enterokinase to restore its active natural structure. Its bactericidal and bacteriostatic activity against avian pahtogenic E.coli and Staphylococcus aureus under various pH values and storage temperatures were tested by determining minimal inhibitory concentration （MIC） and minimal bactericidal concentration （MBC）. Mice model was used to assess its protective effects in vivo. The results showed that MIC and MBC of the recombinant protein against avian pahtogenic E.coli and S.aureus ranged from 1.32 μg/mL to 4.32 μg/mL,and 1.77 μg/mL to 8.54 μg/mL,respectively. The protein is highly stable and could retain a high antimicrobial activity at temperature range of -70 ℃ to 100 ℃ and pH ranges of pH3 to pH10. A dose of 240 μg/mouse of recombinant protein was capable of providing protection against challenge with lethal dose of avian pathogenic E.coli,and 85 % protection rate could be achieved at 320 μg/mouse.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第3期205-209,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
湖南省教育厅科技科研项目(06C406)
湖南省科技厅科技科研项目(06FJ4101)
关键词
重组抗菌肽
Hadrurin
抗菌活性
recombinant antibacterial peptide
Hadrurin
antimicrobial activity
